Purification and properties of an aminoglycoside acetyltransferase from Pseudomonas aeruginosa.
An aminoglycoside 3-acetyltransferase [AAC(3)], possibly a new isoenzymic species of the 3-N-acetyltransferase group, was purified to apparent homogeneity from a crude extract of Pseudomonas aeruginosa, a gentamicin-resistant clinical isolate. The method of purification was consecutive column chromatography--(i) gel filtration, (ii) affinity chromatography, and (iii) ion-exchange chromatography--to give two protein peaks, one of which was coincident with activity and which indicated a purification of 600 (specific activity = 9.743 units mg-1 at pH 7.2, 34 degrees C). Polyacrylamide disc gel electrophoresis indicated a single protein band coincident with enzymic activity. The molecular weight of the enzyme was about 39 000. AAC(3)-V (provisonal designation) was further characterized by stability, substrate, pH, and kinetic studies. The Km was 0.724 microM (sisomicin), and the Vmax was 0.102 mumol min-1 mg-1 (sisomicin) at pH 7.2 and 34 degrees C. Substrate inhibition was exhibited by kanamycin A and tobramycin. Studies showed that enzyme activity was significantly stabilized when preparations contained substrate.
D000123:Acetyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013379:Substrate Specificity
D007527:Isoenzymes; D000123:Acetyltransferases; C019681:aminoglycoside acetyltransferase